Antisense oligonucleotides to type I procollagen

ABSTRACT

The invention provides antisense DNA oligonucleotides which are effective in inhibiting the expression of a wild type COL1A1 gene.

The present invention relates to antisense oligonucleotides and theiruse in inhibiting expression of type I procollagen.

The collagens are a family of closely related proteins, with a triplehelix protein structure. Numerous collagen types have been identified(>10) of which type I procollagen (consisting of two alpha1 chains andone alpha2 chain) is the principal component of bone, skin, and tendon.

It has been recognised for many years that many pathological conditionsare caused by overproduction of collagen fibres in the form of scars andexcess fibrous tissues. For example, liver cirrhosis is a two-stepprocess in which normal liver tissue is first destroyed by a virus or byalcohol and other toxins, and then excessive amounts of collagen fibresreplace the damaged cells before normal liver cell regeneration.Idiopathic pulmonary fibrosis is a lethal condition in which normal lungtissue is gradually replaced by excessive amounts of collagen fibres.Progressive systemic sclerosis (scleroderma) is a frequently lethaldisease where skin and many internal organs become leather-like becauseof excessive depositions of collagen fibres. In many individuals, woundsor surgical incisions in the skin are followed by excessive depositionsof collagen in the form of hypertrophic scars and keloids that presentcosmetic problems and sometimes more serious consequences. Also,excessive scarring frequently occurs in normal individuals followingtrauma and surgical procedures. In these and related conditions, a meansof specifically inhibiting collagen synthesis and deposition would be oftremendous benefit.

PCT Patent Application Publication No. WO 94/11494 discloses a DNA orRNA oligonucleotide comprising from 5 to 200 nucleotides substantiallycomplementary to a mutant collagen nucleotide sequence or a normal wildtype collagen nucleotide sequence which is capable of inhibitingcollagen gene expression. Preferred oligonucleotides are said to beantisense oligonucleotides. The Examples of WO 94/11494 describe aseries of DNA oligonucleotides, some of which are antisense, that weresynthesised primarily with regard to the region at the 3′ end of exon 1(from nucleotides 198 to 222) and the first two nucleotides of intron 1of the human gene for the proα1 chains of type I procollagen (COL1A1).The synthesised oligonucleotides were found to vary considerably intheir ability to inhibit expression of an internally deleted mutantCOL1A1 gene of human origin. The effectiveness of the oligonucleotidesin inhibiting the expression of the human wild type COL1A1 gene was nothowever demonstrated. Since the structure and conformation of the RNAtranscripts of the human, mutant and wild type COL1A1 genes would mostlikely differ, it would not necessarily follow that oligonucleotideswhich are effective inhibitors of the expression of the mutant COL1A1gene would also be effective inhibitors of the expression of the wildtype COL1A1 gene.

It would be desirable to identify antisense DNA oligonucleotides thatare capable of inhibiting the expression of a wild type COL1A1 gene.

In accordance with the present invention, there is therefore provided anantisense DNA oligonucleotide comprising from 18 to 25 nucleotides whichis complementary to a nucleotide sequence from position 750 to position3900 inclusive of SEQ ID NO:1, wherein SEQ ID NO:1 comprises anucleotide sequence encoding a polypeptide comprising an amino acidsequence according to SEQ ID NO:2, the oligonucleotide being capable ofinhibiting expression of the polypeptide in a cell that expresses it.

SEQ ID NO:1 is identical to the nucleotide sequence registered underEMBL accession no. Z74615. SEQ ID NO:2 is the amino acid sequence of thepolypeptide encoded by the nucleotide sequence of SEQ ID NO:1. Thepolypeptide encoded by SEQ ID NO:1 is a precursor of the wild type,proα1 chain of type I procollagen (“prepro-alpha1 (I) collagen”).

The antisense DNA oligonucleotide according to the invention comprises18, 19, 20, 21, 22, 23, 24 or 25 nucleotides and is preferably 20nucleotides in length.

The antisense DNA oligonucleotide is preferably complementary to anucleotide sequence in one of the following regions of SEQ ID NO:1,

Region 1 from position 750 to position 900 inclusive, Region 2 fromposition 1200 to position 1300 inclusive, Region 3 from position 1400 toposition 1500 inclusive, Region 4 from position 1450 to position 1550inclusive, Region 5 from position 1850 to position 2000 inclusive,Region 6 from position 2500 to position 2600 inclusive, Region 7 fromposition 2850 to position 2950 inclusive, Region 8 from position 3800 toposition 3900 inclusive.

Particularly preferred antisense DNA oligonucleotides are those whichare complementary to a nucleotide sequence in Region 2, 4, 6 or 8 of SEQID NO:1.

The oligonucleotides of the invention may be prepared by any suitablemethod known in the art. The oligonucleotides are very convenientlyprepared by synthetic chemical methods, for example, phosphoramiditechemistry by sulfurization with tetraethylthiuram disulfide inacetonitrile as described in Tetrahedron Lett., 1991, 32, 30005–30008.

The oligonucleotides of the present invention are advantageous in thatthey inhibit expression of the wild type COL1A1 gene. They are thereforeuseful in the treatment or prevention of conditions/disorders caused byoverproduction of collagen fibres, for example, liver cirrhosis, kidney,liver and heart fibrosis, scleroderma, hypertrophic scars and keloids.

Accordingly, the present invention provides an antisense DNAoligonucleotide according to the invention for use in therapy.

In another aspect, the invention provides the use of an antisense DNAoligonucleotide according to the invention in the manufacture of amedicament for use in therapy.

In the context of the present specification, the term “therapy” alsoincludes “prophylaxis” unless there are specific indications to thecontrary. The terms “therapeutic” and “therapeutically” should beconstrued accordingly.

The invention further provides a method of treating, or reducing therisk of, a collagen disorder in a patient suffering from, or at risk of,the disorder, which comprises administering to the patient atherapeutically effective amount of an antisense DNA oligonucleotideaccording to the invention.

For the above-mentioned therapeutic uses the dosage administered will,of course, vary with the oligonucleotide employed, the mode ofadministration, the treatment desired and the disorder indicated.Effective dosages are those which are able to inhibit collagen proteinproduction in cells at a level which eliminates or reduces the symptomsor conditions associated with the collagen protein production.

The oligonucleotides according to the invention will generally beadministered in the form of a pharmaceutical composition in which theoligonucleotide is formulated with a pharmaceutically acceptableadjuvant, diluent or carrier.

Thus, the present invention also provides a pharmaceutical compositioncomprising an antisense DNA oligonucleotide according to the inventionin association with a pharmaceutically acceptable adjuvant, diluent orcarrier.

The invention further provides a process for the preparation of apharmaceutical composition of the invention which comprises mixing theantisense DNA oligonucleotide with a pharmaceutically acceptableadjuvant, diluent or carrier.

The pharmaceutical composition of the invention may be administeredtopically in the form of, for example, a creme, lotion or ointment; orsystemically, e.g. by oral administration in the form of tablets,capsules, syrups, powders or granules, or by parenteral administrationin the form of sterile solutions or suspensions.

The present invention will now be further explained by reference to thefollowing illustrative Examples.

EXAMPLES Example 1

Oligonucleotide Synthesis

Phosphorothioate oligodeoxynucleotides synthesis was carried out at a 1μm scale on PE Biosystems 394 DNA synthesizer using phosphoramiditechemistry with TETD/acetonitrile sulphurizing reagent. Oligonucleotideswere purified on Poly-Pak TM II cartridges (Glen Research), desalted onNAPTM 10 columns (Amersham Pharmacia Biotech AB) and ion-exchanged usingDowex 50WX8-1100 ion exchange resin (Aldrich). Twelve antisense DNAoligonucleotides (ASOs) were prepared having the following sequences(5′→3′):

1. GGACGACCAGGTTTTCCAGC (SEQ ID NO:3) 2. GCAGCACCAGCAGGGCCAGG (SEQ IDNO:4) 3. GCCAGGAGCACCAGGTTCAC (SEQ ID NO:5) 4. CTTCCTCTCCAGCAGGGCCA (SEQID NO:6) 5. GCCTTGCCGGGCTCTCCAGC (SEQ ID NO:7) 6. CGGGAACACCTCGCTCTCCA(SEQ ID NO:8) 7. GCAGGACCGACAGCGCCAGG (SEQ ID NO:9) 8.TCCATCTTTGCCAGCAGGAC (SEQ ID NO:10) 9. GGTCCCTGAGCTCCAGCCTC (SEQ IDNO:11) 10. TTGGCCGTCAGCACCAGGG (SEQ ID NO:12) 11. TTTCTCGCCAGCAGGGCCAG(SEQ ID NO:13) 12. CTCGATCTGCTGGCTCAGGC (SEQ ID NO:14)

Example 2

Treatment of Cells

The human cell line WI-26 was grown in Dulbecco's modified Eagle'smedium (DMEM) containing 10% fetal calf serum. The cells were plated in48-well plates or 6-well plates (Costar, Coming Inc.) to obtain 70–80%confluence. After 24 hours, the cells were washed two times withpre-warmed DMEM and 0.35 ml (for 48-well experiments) or 1 ml (6-wellexperiments) DMEM containing 5 μg/ml lipofectin (Gibco BRL) or 2.5 μg/mlcytofectin GSV (Glen Research Ltd) and oligonucleotides at 200 nM wereadded to each well. After 4–5 hours at 37° C. the cells were washed twotimes with pre-warmed DMEM and 0.35 ml DMEM (48-well plates) or 1 mlDMEM (6-well plates) was added together with ascorbic acid at 20 ug/ml.The cells were incubated for 20 hours prior to analysis of collagenlevels.

Example 3

Protein Analysis

At the end of the experiment, 150 μl of medium was removed and theamount of secreted type I procollagen determined using an ELISA kit(AmershamPharmacia Ltd) and the results expressed as nanograms ofprocollagen in the medium/10,000 cells. To correct for cell numbers,plates were washed with pre-warmed PBS, cells treated with trypsin andcell numbers determined using an automated Coulter counter. For 6-wellexperiments, the cells were counted, treated with 1 ml TRI reagent(SIGMA Ltd) and proteins and RNA extracted according to themanufacturers guidelines. The protein pellet was re-suspended in 1% SDScontaining protease inhibitors. 30–100 ugs cellular proteins were heatedat 100° C. for 5 mins and then lectrophoresed in a 4–12% SDSpolyacrylamide gel. Proteins were electrophoretically transferred tonitrocellulose filters and hybridised with an antibody against asynthetic peptide corresponding to human proα1 (I) chain of type 1collagen (obtained from Dr Larry Fisher, NIH, USA). The proα1 (1) bandwas detected using an anti-rabbit secondary antibody coupled to HRP(Biorad Ltd) and developed using ECL (Pierce Ltd). Protein loading wasdetermined by treating the membrane with an antibody to GAPDH (AdvancedImmunochemicals). Protein loading was normalised to GAPDH levels usingdesitometry.

Example 4

RNA Analysis

RNA was extracted using TRI reagent and the final pellet wasre-suspended in 0.5% SDS. One to three micrograms of total RNA wereelectrophoresed in a formaldehyde denaturing gel according to standardprocedures (Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989)Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). RNA was transferred toHybond-N membranes (Amersham) and hybridised for 24 hours to an alpha1(1) cDNA probe radiolabeled using a T7 polymerase kit(AmershamPharmacia). Following washing, the filter was exposed to X-rayfilm and the film developed 4–24 hours later. The autoradiographicimages of the alpha1 (1) transcripts (4.8 kb & 5.8 kb) were analysed bydensitometric analysis and RNA loading was corrected using the intensityof the GAPDH transcript or the intensity of the 28S rRNA as internalcontrols.

Results

Table I below shows the average percentage (%) collagen inhibition whichrelates to either collagen levels in the medium or collagen mRNA levels.In the treated cell assay used, there was a very good correlationbetween percentage collagen inhibition as measured in the medium andpercentage inhibition of intracellular collagen mRNA levels.

TABLE I AVERAGE % COLLAGEN ASO INHIBITION GGACGACCAGGTTTTCCAGC 50 (SEQID NO:3) GCAGCACCAGCAGGGCCAGG 50–80 (SEQ ID NO:4) GCCAGGAGCACCAGGTTCAC50 (SEQ ID NO:5) CTTCCTCTCCAGCAGGGCCA 50–60 (SEQ ID NO:6)GCCTTGCCGGGCTCTCCAGC 50 (SEQ ID NO:7) CGGGAACACCTCGCTCTCCA 50 (SEQ IDNO:8) GCAGGACCGACAGCGCCAGG 50 (SEQ ID NO:9) TCCATCTTTGCCAGCAGGAC 50 (SEQID NO:10) GGTCCCTGAGCTCCAGCCTC 50 (SEQ ID NO:11) TTGGCCGTCAGCACCAGGG50–80 (SEQ ID NO:12) TTTCTCGCCAGCAGGGCCAG 50–70 (SEQ ID NO:13)CTCGATCTGCTGGCTCAGGC 50–80 (SEQ ID NO:14)

1. An antisense DNA oligonucleotide comprising 20–25 nucleotides whichis complementary to SEQ ID NO: 1, wherein SEQ ID NO: 1 comprises anucleotide sequence encoding a polypeptide comprising an amino acidsequence according to SEQ ID NO: 2, the oligonucleotide being capable ofinhibiting expression of the polypeptide in a cell that expresses it, inwhich said oligonucleotide is SEQ ID NO:
 14. 2. A composition comprisingan oligonucleotide as defined in claim 1 in association with apharmaceutically acceptable adjuvant, diluent, or carrier.